The formation of myofibrils is a highly complex process. Initiation of this process is poorly understood. In cultured cardiac muscle cells, stress fiber-like structures first appear, providing the basis for a hypothesis that these structures serve as templateds for further development toward mature myofibrils. The muscle specfic proteins are expressed in the following order in skeletal muscle maturation: desmin, titin, actin, and myosin. In the first phase of the project, we proposed to reveal the organization of the titn network in mature myoflbrlls. This task was very difficult due to the close apposition of this protein to other contractile components of the sarcomere: Using gelsolin, we were able to obtain very clear patterns of titin labeling in the fluorescent microscope. However, the organization of very thin titin filaments was either destroyed or collapsed when some conventional techniques for EM were employed: freeze-drying and evaporation, CPD and coating or thin negative staining. Therefore our attention turned toward IVEM on whole-mounts of myofibrils and thick sections. We used the IvBM to: 1) examine the 3-dimensional arrangement of myoflbrillar proteins in embryonic muscle; 2) determine whether titin and Z-line proteins are associated with small complexes of thick and thin filaments; 3) measure binding of alpha-actinin, filamin and vinculin to isolated myofibrils and skinned fibers; 4) define conditions for assembly of actin into gelsolin treated skinned fibers; 5) identify which peptides of titin are phosphorylated and thier locations in the sarcomere using EM antibody labeling. Some of this work was accomplished at the San Diego NCMIR.